The crude pheromone extracts were prepared according to a previously published protocol (Zhang et al., 2013). N2 wild-type, him-5 mutant, daf-22; him-5 mutant, or TR389 him-5 mutant worms were grown for two generations on 60 mm NGM plate seeded with E. coli OP50 bacteria. Worms from four plates were washed by M9 buffer and cultured in 50 ml S complete medium (100 mM NaCl, 50 mM KPO4, 3 mM CaCl2, 3 mM MgSO4, 5 μg/ml cholesterol, 10 mM potassium citrate, 50 μM disodium EDTA, 25 μM FeSO4, 10 μM MnCl2, 10 μM ZnSO4, 1 μM CuSO4) at 20°C and 200 rpm. The animals were cultured with shaking at 200 rpm for 7 days (around 30,000 worms/50 ml). 25× Concentrated E. coli OP50 bacteria were supplemented every day (0.3 ml for day 1, 1 ml for days 2–5, and 2 ml for days 6–7). After 7 days, the supernatant medium containing metabolites was collected by centrifugation (3000 g, 10 min). Then the supernatant media were frozen at −80°C, lyophilized, and extracted with methanol for UPLC-MS analysis. UPLC-MS was performed using a Sciex TripleTOF 6600 system. Chromatographic separations were achieved using a Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 10 mm), with a flow rate of 0.4 ml/min. Data acquisition and processing were performed by Analyst and Peakview (Sciex).

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